In vivo dynamics of AAV-mediated gene delivery to sensory neurons of the trigeminal ganglia

Figure 1. Comparison of ocular and whiskerpad delivery routes for gene delivery to trigeminal ganglia. Swiss Webster mice were inoculated with PBS or scAAV1-smCBA-mCherry vector at a dose of 1 × 1011 vector genomes per delivery site. For whiskerpad delivery AAV was delivered by intradermal injection in a volume of 50 μl. For ocular delivery AAV was delivered in a volume of 5 μl onto the eye of mice with non-scarified or scarified corneas. At 7 days post AAV delivery DNA was isolated from trigeminal ganglia and total levels of AAV vector genomes per ganglion were quantified by qPCR. Each circle represents a single trigeminal ganglion (TG) from 2 experimental animals (4 TGs total) per route for AAV1 and 1 animal (2 TGs) per route for PBS control.

Figure 2. Comparison of AAV serotypes for gene delivery to trigeminal ganglia. Swiss Webster mice were inoculated by intradermal injection of the whiskerpad with PBS or scAAV-smCBA-GFP vectors packaged into AAV serotypes 1, 5, 7, 8 or 9 at a dose of 1 × 1011 vector genomes per whiskerpad. (a) At 7 days post AAV delivery DNA was isolated from trigeminal ganglia and total levels of AAV vector genomes per ganglion were quantified by qPCR. Each symbol represents one trigeminal ganglion (TG) from a total of either 2 animals for each AAV serotype or 1 animal for PBS control. (b) At 14 days post AAV delivery the percentage of neurons positive for mCherry by immunohistochemistry were quantified in trigeminal ganglia using Cell Profiler software. Each symbol represents the average number of GFP positive neurons from 2 sections of an individual trigeminal ganglion from 3 different animals for each AAV serotype and PBS control. (c) Immunohistochemical detection of GFP and (d) hematoxylin/eosin staining of representative sections from trigeminal ganglia harvested at 14 days post AAV delivery. Scale bar: 100 μm.

Figure 3. Comparison of constitutive and neuronal-specific promoters for gene expression in trigeminal ganglia. Swiss Webster mice were inoculated by intradermal injection of the whiskerpad with PBS or 1 × 1011 vector genomes of scAAV1-CMV-GFP, scAAV1-sCMV-GFP, scAAV1-smCBA-GFP or scAAV1-hSyn-GFP. Sections from trigeminal ganglia harvested at 14 days post AAV delivery were stained for GFP by immunohistochemistry. Scale bar: left panel - 1 mm, right two panels - 100 μm.

Figure 4. Longitudinal kinetics of scAAV1 gene delivery to trigeminal ganglia. Swiss Webster mice were inoculated by intradermal injection of the whiskerpad with PBS or 1 × 1011 vector genomes of scAAV1-smCBA-mCherry. At days 3, 7, 14, 21, and 28 post-delivery trigeminal ganglia were analyzed for levels of vector genomes by qPCR (a) or the percentage of neurons positive for mCherry by immunohistochemistry (b). For qPCR each symbol represents one trigeminal ganglion (TG) from a total of either 2 animals for AAV1 or 1 animal for PBS control at each time point. mCherry positive neurons were quantified using Cell Profiler software and each symbol represents the average number of mCherry positive neurons from 5 sections of one individual trigeminal ganglion (TG) from a total of either 2 animals for AAV1 or 1 animal for PBS control at each time point. Representative sections stained by immunohistochemistry for mCherry (c) or with hematoxylin/eosin (d) are shown from PBS or scAAV1-smCBA-mCherry injected mice. Scale bar: 100 μm.

Figure 5. AAV co-transduction of sensory neurons in the trigeminal ganglia. (a,b) Swiss Webster mice were inoculated by intradermal injection of the whiskerpad with PBS or with 2 × 1011 vector genomes of scAAV1-smCBA-mCherry and 2 × 1011 vector genomes of scAAV1-smCBA-GFP. At 21 days post AAV delivery trigeminal ganglia were harvested and primary explant cultures were established to analyze numbers of mCherry and GFP positive cells by fluorescence microscopy. Representative images of primary sensory neuronal cultures are shown for 3 (a) and 29 days (b) post explant. For quantification GFP and mCherry positive neurons were counted in a total of 10 fields of view for each neuronal culture over 29 days.

Figure 6. Dose optimization of scAAV1 gene delivery to trigeminal ganglia. Swiss Webster mice were inoculated by intradermal injection of the whiskerpad with PBS or increasing doses of scAAV1-smCBA-mCherry. At 6 weeks post AAV delivery trigeminal ganglia were analyzed for levels of vector genomes by qPCR (a). At the two highest doses the percentage of neurons positive for mCherry by immunohistochemistry were also determined (b). For qPCR each symbol represents one trigeminal ganglion (TG) from a total of either 2 animals for AAV1-treatment or 1 animal for PBS control for each dose. mCherry positive neurons were quantified using Cell Profiler software and each symbol represents the average number of mCherry positive neurons from 2 sections of one individual trigeminal ganglion (TG) from 1 animal for each dose. Representative sections stained by immunohistochemistry for mCherry (c) or with hematoxylin/eosin (d) are shown from PBS or scAAV1-smCBA-mCherry injected mice. Representative sections showing NeuN staining (Upper panel) or mCherry staining (Lower panel) in the V1 (Olfactory), V2 (Maxillary) and V3 (Mandibular) branches of the trigeminal ganglion are shown for animals that received 1 ×…